Throughout this application various publications are referred to in short form. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
22q13 deletion syndrome. Chromosome 22q13 deletion syndrome, also known as Phelan-McDermid Syndrome, was first described in case reports in the early 90s, culminating in a review of the 24 published cases and 37 additional cases by Phelan et al. (2001). The studies conclusively demonstrated that individuals identified with 22q13 deletion syndrome had global developmental delay and absent or severely delayed expressive speech. Furthermore, the overwhelming majority of cases had hypotonia (97%) with normal or accelerated growth (95%). The developmental delay is associated with mental retardation typically in the mild-to-moderate range. Other, less universal, features included large hands (>75%), dysplastic toenails (>75%), and decreased perspiration. Behavior characteristics include mouthing or chewing non-food items (>75%), decreased perception of pain (>75%), and autism or autistic-like traits. Approximately 75% of individuals with a 22q13 deletion syndrome diagnosis have either a 22 q terminal deletion (i.e., a chromosome break in 22q with loss of the segment distal to the break), or an interstitial deletion (i.e., two breaks within the same chromosome arm and loss of the intervening segment). The remaining 25% of individuals diagnosed with 22q13.3 deletion syndrome had deletions resulting from an unbalanced translocation or other structural rearrangement, including ring 22.
Hypotonia, global developmental delay and speech deficits together represent some of the most consistent findings, each in >95% of all patients. The hypotonia in newborns with the syndrome can be associated with weak cry, poor head control, and feeding difficulties leading to failure to thrive. In terms of developmental delay, in addition to the mental retardation noted above, there is also evidence for a delay to major milestones, such that, for example, the average age for rolling over is approximately eight months, for crawling approximately 16 months, and for walking approximately three years. Poor muscle tone, lack of balance, and decreased upper body strength contribute to the delay in walking and ultimately, gait is often broad-based and unsteady. Finally, while infants with the syndrome typically babble at the appropriate age and children may acquire a limited vocabulary, by approximately age four years many children have significant deficits in the ability to speak. With intensive therapy, the individuals with the syndrome may have some speech and increase their vocabularies. It is interesting to note that receptive communication skills are more advanced than expressive language skills as demonstrated by the ability of affected children to follow simple commands, demonstrate humor, and express emotions.
Role of SHANK3 in 2203 deletion syndrome. Three lines of evidence implicated a single gene, SHANK3 (for SH3 and multiple ankyrin repeat domains 3, also referred to as proline-rich synapse associated protein 2/PROSAP2), in 22q13 deletion syndrome. First, careful analysis of the extent of the deletion in independent cases indicated a small critical region encompassing SHANK3. Thus, an analysis of 33 cases with various forms of monosomy of chromosome 22 (include ring 22, which as noted above is phenotypical similarly to the deletion syndrome) showed that the 12 with simple deletions had deletions of variable in size (from 160 kb to 9 Mb), with a minimal critical region responsible for the phenotype including SHANK3, ACR, and RABL2B (Luciani et al., 2003). Similarly, an analysis of 56 patients with the syndrome again demonstrated a very variable size of the deletion (130 kb to 9 Mb) with deletion of SHANK3 found in all cases explicitly tested, including the smallest deletion, with the minimal region encompassing the same three genes (Wilson et al., 2003). Remarkably, the severity of the behavioral phenotype was not correlated with the size of the deletion, indicating that haploinsufficiency of just one or more of these three genes was primarily responsible for the phenotype. Higher resolution studies have now identified patients with even smaller deletions, which exclude ACR and RABL2B from the minimal region, leaving only SHANK3 as the causal gene for the deletion syndrome (Bonaglia et al., 2011).
The second line of evidence was the demonstration of a recurrent breakpoint in SHANK3 in some cases with 22q13 deletion syndrome. The first report of a translocation with a breakpoint in SHANK3 associated with 22q13 deletion syndrome already made the point that disruption of SHANK3 likely underlied the disorder (Bonaglia et al., 2001). This group went on the identify two additional cases (Bonaglia et al., 2006), both with a breakpoint within the same 15-bp repeat unit in the SHANK3 gene (which overlapped with another SHANK3 breakpoint described by Wong et al., 1997). The presence of recurrent disruptions in SHANK3 led to the conclusion that disruption of this one gene is sufficient for the generation of 22q13 deletion syndrome.
Role of SHANK3 in autism spectrum disorders (ASD). Mutations directly in SHANK3 also resulting in the main features of 22q13 deletion syndrome represent the final line of evidence. Thus, while it has become increasingly recognized that 22q13 deletion syndrome can present with ASD and in fact 22q13 deletions are commonly associated with ASD in literature surveys (Vorstman et al., 2006), three recent studies explored the separate question as to whether SHANK3 disruption and mutations can be found in cohorts with apparently idiopathic ASD. In the first such study (Durand et al., 2007), SHANK3 was analyzed by both FISH and by direct sequencing in as many as 227 individuals with ASD. Three variants were identified. First, an individual with a de novo deletion of SHANK3 was identified; this individual had autism (narrowly defined), absent language, and moderate mental retardation. Second, a paternally inherited translocation was identified that resulted in a deletion of the 22q13 region (including SHANK3) in a girl with autism and severe language delay, and a duplication of the same region in her brother with Asperger syndrome. Finally, Durand et al. (2007) identified two brothers with autism, severely impaired speech, and severe mental retardation, which carried a single-base insertion in SHANK3. The insertion, which was maternal in origin (likely due to germline mosaicism in the mother), resulted in a frameshift at the COOH-terminal of the protein that disrupts domains involved in Homer and cortactin binding and the sterile alpha motif (SAM) domain involved in assembly of the SHANK3 platform. Overexpression of the mutant form in cultured hippocampal neurons did not lead to synaptic localization of the heterologous protein, in contrast to the wild-type SHANK3 protein.
In a follow up to Durand et al. (2007), Moessner et al. (2007), examined both sequence and SHANK3 gene dosage in 400 individuals with ASD. Two deletions were identified, as well as 1 de novo mutation. Furthermore, an additional deletion was identified in two siblings from an additional collection. The mutation, found in a girl with autism, results in a Q321R change in the ankyrin repeat domain at the NH2 terminal of SHANK3.
In a third study, Gauthier et al. (2009) sequenced SHANK3 in 427 ASD subjects and identified a de novo deletion at an intronic donor splice site and a missense variant transmitted from an epileptic father.
A de novo splice site variant of the SHANK3 gene has also been reported in a patient with mental retardation and severe language delay (Hamdan et al., 2011). In addition, Shank3 mutant mice display autistic-like behaviours (Bozdagi et al. 2010; Bangash et al., 2011; Peca et al., 2011; Wang et al., 2011).
Remarkably, SHANK3 mutations can also result in schizophrenia, including atypical schizophrenia associated with mental retardation and/or early onset as recently shown by Gauthier et al. (2010).
Altogether, these studies strongly support a role for disruptions of SHANK3 in developmental delay and ASD. Clearly, haploinsufficiency of SHANK3, caused either by a chromosomal abnormality or a mutation, can result in a profound phenotype. Furthermore, even overexpression of SHANK3 can result in developmental disorders (considering, for example, the case with Asperger syndrome and three copies of the SHANK3 locus reported in Durand et al., 2007 or the case with three copies and ADHD reported in Moessner et al., 2007). Recent, very large scale studies in clinical samples demonstrate that ca. 0.3% of patients with intellectual disability referred to for chromosome microarray have a SHANK3 deletion or duplication (Cooper et al., 2011). With the advent of clinical sequencing, point mutations in SHANK3 are also being identified in the clinical setting and evidence from research studies indicates a similar rate (ca. 0.3%) making SHANK3 deletions and mutations one of the more common monogenic causes of developmental delay syndromes, intellectual disability and ASD.
Function of SHANK proteins in the structure of the synapse. The post-synaptic density (PSD) is an electron-dense structure underlying the postsynaptic membrane in glutamatergic synapses in the central nervous system (Okabe, 2007). The PSD is most commonly found on dendritic spines of pyramidal neurons of the neocortex and hippocampus and Purkinje cells of the cerebellum, as well as on dendritic shafts at sites of contact with interneurons in the neocortex and hippocampus, as well as motoneurons in the spinal cord. As such the PSD represents a critical organelle for glutamatergic transmission. It has been shown that the SHANK proteins (including SHANK3) are a major part of the PSD. Multiple analytical approaches, including the characterization of antibodies directed against PSD preparations, two-hybrid screens, gel electrophoresis and mass spectrometry and other modern proteomic approaches have placed the SHANK proteins in the PSD (reviewed in Boeckers, 2006 and Okabe, 2007). Moreover, recent quantitative methods have estimated that there are about 300 individual SHANK molecules in a single postsynaptic site, representing something in the order of 5% of the total protein molecules and total protein mass in the site (Sugiyama et al., 2005). As it has been postulated that SHANK proteins may nucleate the protein framework for the PSD, a recent study examined the ability of the sterile alpha motif (SAM) of SHANK3 to form polymers by self-association (Baron et al., 2006). As with other SAM domains (Qiao and Bowie, 2005), the SAM domain of SHANK3 was able to self-associate, giving rise to large sheets of parallel fibers. These studies support the hypothesis that sheets of the SHANK proteins can form the scaffold or platform onto which the PSD is constructed. Such a role for the SHANK proteins has led to them being called “master scaffolding proteins” of the PSD.
The SHANK protein interactome. With the SHANK proteins (including SHANK3) forming a molecular platform onto which the PSD protein complex can be constructed, other proteins and protein complexes of the PSD can associate with the SHANK platform. Of the various protein complexes associated with glutamatergic synapses, there is good evidence that the NMDA receptor complex (NRC), the metabotropic glutamate receptor complex (mGC), and the AMPA receptor complex (ARC) associate with the SHANK platform (see Boeckers, 2006).
The NRC (Husi et al., 2000), analyzed after isolation by affinity purification, includes receptors, scaffolding proteins, signaling proteins, and cytoskeletal proteins. Amongst the scaffolding proteins identified in the NRC are the SHANK proteins, and it is thought that NMDA receptors are anchored to the SHANK platform through the mediation of PSD-95 and SAPAP/GKAP (see Boeckers, 2006). Thus, NMDA receptors are tethered to the postsynaptic membrane by interaction with PDZ domains of PSD-95, while the guanylate kinase domain of PSD-95 interacts with the SAPAP/GKAP proteins, which in turn bind to the SHANK proteins.
Similarly, mGC is linked to the SHANK platform, at least in part via Homer. The mGC (Farr et al., 2004), analyzed after immunoisolation of mGluR5 and associated molecules, includes SHANK and Homer proteins, both of which have been previously associated with metabotropic glutamate receptors using other methods. Homer proteins bind the cytoplasmic domain of mGlu receptors (Brakeman et al., 1997) and couple mGlu receptors—and hence the mGC—to the SHANK platform (Tu et al., 1999). As SHANK proteins are able to bind to the IP3 receptor, this interaction also links mGlu receptors to the IP3 receptor (Sala et al., 2005).
Finally, the components of the ARC are bound to the SHANK platform. There is evidence for a direct interaction between the GluR1 AMPA receptor and SHANK3 (Uchino et al., 2006). Moreover, there is evidence for an indirect interaction in which transmembrane AMPA regulatory protein (TARP) subunits, including stargazin, bind both AMPA receptors and PSD-95 (e.g., Bats et al., 2007). The interaction of AMPA receptors with PSD-95 in turn allows for the linking of AMPA receptors with the SHANK platform via SAPAP/GKAP.
There are additional important interactions that involve the SHANK platform, but even focusing on these three protein complexes, NRC, mGC, and ARC, it is clear that the SHANK proteins are critically involved in the molecular architecture of glutamatergic synapses. Moreover, as SHANK proteins also interact with F-actin (the major cytoskeletal component of spines) through cortactin (Naisbitt et al., 1999) and additional mechanisms (see Boeckers, 2006), the SHANK platform is also likely involved in the dynamic remodeling of glutamatergic synapses over short and longer time frames (e.g., Hering and Sheng, 2003).
Modulation of SHANK3 expression and synapse formation. Overexpression of SHANK1 leads to increased spine size in neurons in culture (Sala et al., 2001). This effect, which could be further enhanced with the cotransfection of Homer 1, also led to the recruitment of Homer, PSD-95, and GKAP to the spines, along with glutamate receptors, the 1P3 receptor, and F-actin and bassoon, with enhancement of synaptic function, as measured electrophysiologically (Sala et al., 2001). More recent studies with SHANK3 support these conclusions (Roussignol et al., 2005). Thus, introduction of an siRNA construct inhibiting SHANK3 expression led to reduced number of spines in hippocampal neurons in culture. Furthermore, Roussignol et al. (2005) demonstrated that the introduction of SHANK3 into aspiny cerebellar neurons was sufficient to induce functional dendritic spines in these cells, which then express functional NMDA and AMPA receptors. Altogether, these studies in cultured cells support a critical role for SHANK proteins in the development and function of the PSD and the glutamatergic synapse.
Recently, SHANK1 homozygous knockout mice were described which showed alterations in PSD thickness and PSD protein make-up, changes in spine morphology, and decrease glutamatergic synaptic strength (but no changes in long term potentiation (LTP)) (Hung et al. 2008). These changes were associated with an increase in anxiety behavior, deficiencies on rotarod, impaired memory in a contextual fear task and in retention in a radial maze, but increased acquisition in the radial maze, confirming a role for SHANK proteins in glutamatergic transmission and behavior.
Regulation of SHANK3 expression by methylation. Proper expression of SHANK3 is an important element of spine formation and brain development. Methylation of genes is one important means of regulating expression. Interestingly, in a genome-wide analysis, SHANK3 was identified as one of several genes where there was a clear relationship between methylation status at CpG islands in the gene and expression (Ching et al., 2005). The authors demonstrated that SHANK3 is expressed in brain tissue, where the gene is predominantly unmethylated, and not expressed in lymphocytes, where the CpG island studied in the SHANK3 gene was nearly completely methylated.
The study of Ching et al. (2005) was followed by a more recent study that looked in greater detail at SHANK3 as well as at the CpG islands in SHANK1 and SHANK2 (Beni et al., 2007). The authors identified 5 CpG islands in SHANK3 (one of which—identified by Beni et al. (2007) as CpG 4—was the CpG island studied by Ching et al., 2005) and an equivalent number in SHANK1 and SHANK2. Only SHANK3 demonstrated tissue-specific methylation of CpG islands, with a relationship between methylation and tissue-specific expression. These studies demonstrated not only that methylation at several of the CpG islands of SHANK3 correlated with SHANK3 expression, but also that modulating the methylation of SHANK3 in cells in culture altered SHANK3 expression. Thus, treating primary neuronal cultures with methionine to increase methylation resulted in decreased expression of SHANK3, while treating HeLa cells with the demethylating agent 5-AdC resulted in decreased methylation of SHANK3 and increased expression of this gene in these cells, which do not normally express SHANK3. Significantly, the decreased expression of SHANK3 in primary neurons treated with methionine was associated with decreased numbers of dendritic spines and with decreased spine width, similar to what was observed by this same group with siRNA treatment of such cells (see above and Roussignol et al., 2005).
It has been shown that a proportion (0.5-1%) of children diagnosed with autism or autism spectrum disorders have deletions, duplications or mutations in SHANK3. While individuals with a diagnosis of 22q13 deletion syndrome are relatively rare, autism and autism spectrum disorders occur with a frequency of about 1 in 100 children. Considering this, as well as the rates of intellectual disability syndromes in the population, it can be estimated that at least 1/6,000- 1/16,000 individuals will have deletions, duplications or mutations in SHANK3 with associated phenotypes. This translates to ˜20-60,000 individuals in the USA alone with life-long disability due to alterations in SHANK3 expression. Thus, there is a compelling need for treatments for subjects with 22q13 deletions or duplications or SHANK3 mutations. The present invention addresses this need.